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PubMed2001

The effect of trauma on neutrophil L-selectin expression and sL-selectin serum levels.

Among identified adhesion molecules, the L-selectin on neutrophils enables the first step of leukocyte adherence to activated endothelial cells. To allow firm adhesion of neutrophils, L-selectin is then split off the cell membrane. It was hypothetized that an increase of the constitutively high serum level of soluble L-selectin may indicate an ongoing pathological neutrophil sequestration to the endothelial cells associated with activation and injury of the cells. To evaluate this hypothesis, sL-selectin serum levels and neutrophil L-selectin expression of healthy volunteers (group A, n = 15), as well as of surgical patients, were investigated. Group B (n = 26) included patients subjected to elective limb surgery (mean operation time, 122 min), and group C (n = 45) comprised trauma patients. sL-selectin serum levels were measured daily over a 14-day period. Neutrophil L-selectin expression was evaluated by FACS analysis using the humanized anti-L-selectin antibody HuDreg 55 over a period of 3 days at minimum in both experimental groups. The binding of sL-selectin to endothelial cells was also examined in vitro. Elective limb surgery resulted in lower pre- and post-operative sL-selectin plasma levels (800-1,000 ng/mL) compared to healthy volunteers (1,100-1,200 ng/mL) with insignificant changes throughout the study period. Trauma patients revealed even lower sL-selectin levels (400-600 ng/mL). When these patients were discriminated by the multiple organ dysfunction (MOD) score of Moore in +MOD (n = 9, ISS = 31.7) and -MOD (n = 36, ISS = 25.0), a significant difference became evident. In +MOD patients sL-selectin levels remained on a low basis of 350 ng/mL, whereas in -MOD patients the initial low sL-selectin level subsequently rose to 800 ng/mL, similar to that of elective surgery patients. FACS analysis revealed a significant drop in neutrophil L-selectin expression 24 h after trauma compared to normal. Also, +MOD and -MOD patients were significantly discriminated by the L-selectin expression at this time. The in vitro studies revealed evidence for binding of sL-selectin to endothelial cells independently on the presence of neutrophils. According to our data, increasing severity of the post-operative/posttraumatic course is associated with decreasing sL-selectin serum levels and also reduced neutrophil L-selectin expression. In view of the in vitro results, this probably indicates competitive enhanced binding of sL-selectin to endothelial cells, thus masking the elevated activation of neutrophils and their ability for endothelial adherence.

Adolescent↗
PubMed2005

Contrasting effects of P-selectin and E-selectin on the differentiation of murine hematopoietic progenitor cells.

OBJECTIVE: The two endothelial selectins, P- and E-selectin, are critically important for adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. MATERIALS AND METHODS: Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed 6 months posttransplant. Bone marrow lineage-negative (Lin(-)) Sca-1(+)c-kit(+) cells were then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoietic potential was assessed using in vitro phenotyping and colony-forming assays and in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. RESULTS: Long-term competitive repopulating HSCs were Lin(-)c-kit(bright) and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of Lin(-)Sca-1(+)c-kit(+) cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S(12) compared to the other cultures. In contrast, E-selectin enhanced the differentiation of Lin(-)Sca-1(+)c-kit(+) cells into cells that expressed the granulocyte maturation marker, Gr-1, accompanied by loss of CFC potential from these cultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. CONCLUSION: These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S(12) in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of Lin(-)Sca-1(+)c-kit(+) cells, allowing enhanced ex vivo expansion of CFC and CFU-S(12) but not HSCs.

Animals↗
PubMed1996

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells.

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L-selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino-terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L-selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.

Antigens, CD34↗
PubMed1997

The association between alpha4-integrin, P-selectin, and E-selectin in an allergic model of inflammation.

In this study, we examined the relationship between the endothelial selectins (P-selectin and E-selectin) and whether they are critical for alpha4-integrin-dependent leukocyte recruitment in inflamed (late phase response), cremasteric postcapillary venules. Animals were systemically sensitized and 2 wk later challenged intrascrotally with chicken ovalbumin. Leukocyte rolling flux, adhesion, and emigration were assessed at baseline and 4 and 8 h postantigen challenge. There was a significant increase in leukocyte rolling flux, adhesion, and emigration in sensitized and challenged mice at both 4 and 8 h. At 8 h, the increase in leukocyte rolling flux was approximately 50% inhibitable by an anti-alpha4-integrin antibody, 98% inhibitable by fucoidin (a selectin-binding carbohydrate), and 100% inhibitable by an anti-P-selectin antibody. P-selectin-deficient animals displayed no leukocyte rolling or adhesion at 8 h after challenge. However, at 8 h there were many emigrated leukocytes in the perivascular space suggesting P-selectin-independent rolling at an earlier time point. Indeed, at 4 h postantigen challenge in P-selectin-deficient mice, there was increased leukocyte rolling, adhesion, and emigration. The rolling in the P-selectin-deficient mice at 4 h was largely alpha4-integrin dependent. However, there was an essential E-selectin-dependent component inasmuch as an anti-E-selectin antibody completely reversed the rolling, and in E-selectin and P-selectin double deficient mice rolling, adhesion and emigration were completely absent. These results illustrate that P-selectin underlies all of the antigen-induced rolling with a brief transient contribution from E-selectin in the P-selectin-deficient animals. Finally, the antigen-induced alpha4-integrin-mediated leukocyte recruitment is entirely dependent upon endothelial selectins.

Animals↗
PubMed1997

Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1.

We compared the abilities of selectins and the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), to support tethering and rolling of eosinophils and neutrophils under physiologic flow conditions. Eosinophils and neutrophils accumulated on P-selectin at similar site densities and rolled at similar velocities, but fewer eosinophils than neutrophils accumulated at any P-selectin density. Compared with neutrophils, few eosinophils accumulated on E-selectin except at high densities, and those cells that did accumulate rolled faster than neutrophils. Examination of the mechanisms for accumulation revealed that eosinophils and neutrophils formed similar numbers of primary tethers to P-selectin, whereas eosinophils formed fewer primary tethers to E-selectin than did neutrophils. Compared with neutrophils, adherent eosinophils also supported fewer leukocyte-leukocyte interactions, resulting in diminished secondary tethers to either P- or E-selectin. Studies with mAbs to L-selectin and PSGL-1 demonstrated that neither cell type used L-selectin to form primary tethers to P- or E-selectin. Both eosinophils and neutrophils used the NH2 terminus of PSGL-1 to form primary tethers to P-selectin, but not to E-selectin. Both cell types used L-selectin and PSGL-1 to promote leukocyte-leukocyte interactions and secondary tethers to P- or E-selectin. However, eosinophils developed significantly fewer secondary interactions, probably because they express less L-selectin than do neutrophils.

Cell Adhesion↗
PubMed1997

Blocking both E-selectin and P-selectin inhibits endotoxin-induced leukocyte infiltration into the eye.

The initial contact between leukocytes and the vascular endothelium at sites of inflammation is mediated by selectins. The purpose of this study was to investigate the role of the two selectins expressed on the vascular endothelium, E-selectin and P-selectin, in the pathogenesis of endotoxin-induced uveitis. Endotoxin-induced uveitis was produced in female C3H/HeN mice using Salmonella typhimurium endotoxin injected into one hind footpad. At the time of endotoxin injection mice were treated with an intraperitoneal injection of a monoclonal antibody against E-selectin or P-selectin, a combination of both anti-selectin antibodies, or isotype-matched control antibodies. In a second set of experiments, antibody treatment was administered 6 hr after endotoxin injection, when inflammatory cells are already entering the eye. Ocular inflammation was graded histologically by a masked observer. When administered at the time of endotoxin injection, anti-P-selectin antibody decreased ocular inflammation by 37% compared to control animals (P = 0.05). There was no statistical decrease in ocular inflammation in animals treated with anti-E-selectin antibody. The combination of anti-P-selectin and anti-E-selectin antibodies decreased infiltrating inflammatory cells by 61% (P < 0.01). When treatment was delayed until 6 hr after endotoxin injection, the combination of anti-P-selectin and anti-E-selectin antibodies again decreased ocular inflammation by 60% (P < 0.01). Immunohistochemical staining showed decreased ICAM-1 expression in the eyes of animals treated with the combination of anti-P-and anti-E-selectin antibodies. Blocking both P-selectin and E-selectin resulted in a significant decrease in endotoxin-induced intraocular inflammation.

Animals↗
PubMed2003

P-selectin glycoprotein ligand-1 mediates L-selectin-dependent leukocyte rolling in venules.

Leukocyte rolling in postcapillary venules of inflamed tissues is reduced in L-selectin-deficient mice and mice treated with L-selectin blocking antibodies, but the glycoprotein ligand for L-selectin in inflamed venules is unknown. Here, we show that L-selectin-dependent rolling after P-selectin blockade is completely absent in P-selectin glycoprotein ligand-1 (PSGL-1)-/- mice or wild-type mice treated with a PSGL-1 blocking monoclonal antibody. Immunohistochemistry and flow cytometry failed to show PSGL-1 expression on resting or inflamed endothelium or on platelets. To investigate whether leukocyte-expressed PSGL-1 is mediating L-selectin-dependent rolling, we reconstituted lethally irradiated wild-type mice with PSGL-1-/- bone marrow cells. These chimeric mice showed no L-selectin-dependent rolling, suggesting that leukocyte-expressed PSGL-1 mediates L-selectin-dependent rolling. Frame-to-frame video analysis of L-selectin-dependent rolling in wild-type mice showed that the majority of observed L-selectin-dependent leukocyte rolling was between free flowing leukocytes and already adherent leukocytes or possibly leukocyte fragments, followed by E-selectin-dependent leukocyte rolling along the endothelium. Leukocyte rolling was significantly slower for leukocyte-endothelial than leukocyte-leukocyte interactions. We conclude that leukocyte-expressed PSGL-1 serves as the main L-selectin ligand in inflamed postcapillary venules. L-selectin binding to PSGL-1 initiates tethering events that enable L-selectin-independent leukocyte-endothelial interactions. These findings provide a molecular mechanism for the inflammatory defects seen in L-selectin-deficient mice.

Animals↗
PubMed1992

Cloning of the mouse endothelial selectins. Expression of both E- and P-selectin is inducible by tumor necrosis factor alpha.

E-selectin (ELAM-1) and P-selectin (GMP-140, PADGEM, CD62) have both been described as human endothelial cell adhesion molecules for neutrophils and monocytes. Cell surface appearance of these two proteins on human umbilical vein endothelial cells can be regulated by different mechanisms: E-selectin is transcriptionally induced, within hours, by tumor necrosis factor alpha (TNF-alpha) while P-selectin is transported from storage granules to the plasma membrane within minutes upon induction by various stimulating agents. We have obtained cDNA clones covering the full-length coding sequence of the homologous mouse proteins for both endothelial selectins. We show that synthesis of mouse P-selectin, like that of E-selectin, is transiently induced by TNF-alpha in several endothelioma cell lines derived from different mouse tissues. This induction was monitored on both the RNA as well as the protein level. The TNF induced, newly synthesized P-selectin protein was detected on the cell surface. Protein expression of P-selectin increased with a slightly lower rate than expression of E-selectin. In organ cultures of lung tissue from TNF-injected mice, the synthesis of P-selectin was clearly elevated compared to control mice. The bovine P-selectin homolog recognized by cross-reaction with anti-mouse P-selectin antibodies was also TNF-inducible in primary capillary endothelial cells from adrenal cortex and in aorta-derived endothelial cells. Thus, P-selectin can be regulated on two different levels. While the transport of stored P-selectin to the cell surface is controlled by regulated secretion of storage granules, the synthesis and surface expression can be induced by TNF-alpha similarly to that of E-selectin.

Amino Acid Sequence↗
PubMed1995

Activation of polymorphonuclear leukocytes reduces their adhesion to P-selectin and causes redistribution of ligands for P-selectin on their surfaces.

In acute inflammatory responses, selectins mediate initial rolling of neutrophils (PMNs) along the endothelial surface. This is followed by tight adhesion that requires activation-dependent up-regulation of CD11/CD18 integrins on PMNs. For emigration to occur, the initial bonds that are established at the endothelial surface must be disengaged. We show that activation of PMNs results in their detachment from P-selectin, a glycoprotein expressed at the surface of inflamed endothelium that mediates initial tethering of PMNs. Loosening of the bond occurs when PMNs are activated by platelet-activating factor, which is coexpressed with P-selectin, or by other signaling molecules. The time course of reduced adhesion to P-selectin, when compared to up-regulation of CD11/CD18 integrins, suggests that "bond trading" may occur as activated PMNs transmigrate in vivo. Activation of PMNs did not alter binding of fluid-phase P-selectin, indicating that the ligand(s) for P-selectin is not shed or internalized. Using microspheres coated with P-selectin, we found that ligands for P-selectin were randomly distributed over the surfaces of rounded, unactivated PMNs. An antibody against P-selectin glycoprotein ligand-1 (PSGL-1) completely inhibited binding of P-selectin-coated beads suggesting that P-selectin glycoprotein ligand-1 is the critical binding site in this assay. In contrast to the dispersed pattern on unactivated PMNs, the ligands for P-selectin were localized on the uropods of activated, polarized cells. Pretreating PMNs with cytochalasin D before activation prevented the change in cell shape, the redistribution of binding sites for P-selectin-coated beads, and the decrease in cellular adhesiveness for P-selectin. These experiments indicate that the distribution of ligands for P-selectin is influenced by cellular activation and by cytoskeletal interactions, and that redistribution of these ligands may influence adhesive interactions. Activation of PMNs may cause loosening or disengagement of bonds between P-selectin and its ligands, facilitating transendothelial migration.

Animals↗
PubMed1997

The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated.

The HECA452 carbohydrate epitope, also termed cutaneous lymphocyte antigen, is known to bind to E-selectin and defines a human T cell subset preferentially found in inflamed skin. Activated T cells can express a functional form of the P-selectin glycoprotein ligand-1 (PSGL-1), the major ligand known for P-selectin. Here we show that PSGL-1 can exist in two forms, of which only one carries the HECA452 epitope and binds to E-selectin, while the other only binds to P-selectin. We have analyzed the glycoprotein ligands for E- and P-selectin on the mouse CD8+ T cell clone 4G3 at 4, 8, and 12 days after antigen-specific activation. Only at day 4 did the cells bind to E-selectin, whereas cells at all three activation stages bound to P-selectin. Expression of the HECA452 epitope correlated with E-selectin binding. In affinity isolation experiments, PSGL-1 was isolated as the major ligand by E-selectin-IgG and by P-selectin-IgG; however, PSGL-1 only bound to E-selectin at day 4, whereas it bound to P-selectin at all three activation stages. Immunoprecipitated PSGL-1 from cells at day 4, but not from cells at days 8 and 12, was recognized in immunoblots by monoclonal antibody HECA452. In immunoblots of total extracts of cells at day 4, HECA452 recognized a 240/140-kDa pair of protein bands as the major antigen. These bands could be completely removed by depletion of cell extracts with anti-PSGL-1 antibodies. Our data suggest that the carbohydrate requirements for binding of PSGL-1 to P-selectin differ from those necessary for binding to E-selectin. Furthermore, we conclude that the major glycoprotein carrier for the HECA452 epitope on activated 4G3 cells is PSGL-1.

Animals↗
PubMed1996

Interactions of human alpha/beta and gamma/delta T lymphocyte subsets in shear flow with E-selectin and P-selectin.

We have compared the ability of human alpha/beta and gamma/delta T lymphocytes to adhere to selectin-bearing substrates, an interaction thought to be essential for homing and localization at sites of inflammation. Both T cell populations form rolling adhesions on E- and P-selectin substrates under physiologic flow conditions. Although equivalent to alpha/beta T cells in binding to E-selectin, gamma/delta T cells demonstrated greater ability to adhere to P-selectin that was purified or expressed on the surface of activated, adherent platelets. Under static conditions, 80% of gamma/delta T cells and 53% of alpha/beta T cells formed shear-resistant adhesions to P-selectin, whereas only 30% of gamma/delta and alpha/beta T cells adhered to E-selectin. The enhance ability of gamma/delta T cells to adhere to P-selectin cannot be attributed to differences in expression of the P-selectin glycoprotein ligand (PSGL-1), as all alpha/beta T cells versus approximately 75% of gamma/delta T cells expressed PSGL-1. Both cell populations expressed a similar percentage of the carbohydrate antigens sialyl LewisX and cutaneous lymphocyte-associated antigen. Depletion of lymphocyte populations or T cell clones bearing these oligosaccharides with the monoclonal antibody CSLEX-1 and HECA-452, respectively, resulted in a substantial reduction in adhesion to E-selectin and slight reduction in adhesion to P-selectin under flow conditions. Treatment of cells with an endopeptidase that selectively degrades O-sialomucins such as PSGL-1, abolished P-selectin but not E-selectin adhesion. Removal of terminal sialic acids with neuraminidase or protease treatment of cells abrogated cell adhesion to both selectin substrates. These results provide direct evidence for the presence of distinct E- and P-selectin ligands on T lymphocytes and suggest that gamma/delta T cells may be preferentially recruited to inflammatory sites during the early stages of an immune response when P-selectin is upregulated.

Antigens, CD↗
PubMed2000

Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment.

Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment.

Cell Adhesion↗
PubMed2002

Up-regulation of L-selectin and E-selectin in hypersensitivity pneumonitis.

BACKGROUND: Selectins are adhesion molecules that contribute to leukocyte recruitment into the tissue after an injury. Hypersensitivity pneumonitis (HP) is a lymphocytic alveolitis, and we hypothesized that the overexpression of selectins could play a role in this process. PATIENTS AND MEASUREMENTS: We studied 16 patients with HP and 7 healthy control subjects (HCs). Sera and BAL selectins and tumor necrosis factor-alpha were determined by enzyme-linked immunosorbent assay, and cellular lung localization was determined by immunohistochemistry. Additionally, BAL L-selectin, and L-selectin-bearing T-lymphocytes analyzed by flow cytometry were evaluated in HP patients and in exposed but asymptomatic subjects (EAS). SETTING: Tertiary referral center and immunohistochemistry laboratory. RESULTS: Raised levels of E-selectin (mean [+/- SD], 178.9 +/- 30.5 vs 59.4 +/- 4.7 ng/mL, respectively; p < 0.001) and P-selectin (mean, 232.6 +/- 29.9 vs 67.6 +/- 14.2 ng/mL, respectively; p < 0.001) were detected in HP patient sera compared to control subjects, while L-selectin levels showed no differences between groups. Conversely, HP patients displayed a significant increase in levels of L-selectin found in BAL fluid compared with both HCs and EAS (11.0 +/- 1.7 vs 6.9 +/- 0.43 and 3.1 +/- 0.5 ng/mL, respectively; p < 0.05). The levels of E-selectin found in BAL fluid were similar in patients from both groups, and P-selectin was not detected. Percentage of CD3+CD62 L+ lymphocytes was lower in HP patients compared with EAS (2.33 +/- 0.8 vs 4.31 +/- 2.4, respectively; p = 0.05). By immunohistochemistry, L-selectin was detected in interstitial macrophages and polymorphonuclear cells, and E-selectin was detected in endothelial cells. CONCLUSION: These findings demonstrate that L-selectin and E-selectin are up-regulated during the development of HP, suggesting that they may contribute to the increased traffic of lung inflammatory cells.

Adult↗
PubMed1999

Overlapping roles for L-selectin and P-selectin in antigen-induced immune responses in the microvasculature.

Although L-selectin mediates lymphocyte attachment to endothelial venules of peripheral lymph nodes, its role in leukocyte recruitment into tissues following Ag challenge is less well established. The objective of this study was to systematically examine the role of L-selectin in leukocyte rolling in the peripheral microvasculature during the first 24 h of an immune response. A type I hypersensitivity response was elicited in wild-type (C57BL/6) and L-selectin-deficient mice by systemic (i.p.) sensitization and intrascrotal challenge with chicken egg OVA. The cremaster microcirculation was observed in untreated and sensitized mice 4, 8, and 24 h post-Ag challenge by intravital microscopy. Leukocyte recruitment in L-selectin-deficient mice and wild-type mice treated with an L-selectin function-blocking mAb was examined at each time point. Ag challenge induced a significant increase in leukocyte rolling (60 cells/min/venule to approximately 300 cells/min/venule) in wild-type mice at 4-24 h. This response was reduced by approximately 60-70% in L-selectin-deficient mice and in wild-type mice treated with an L-selectin-blocking mAb. P-selectin blockade by Ab completely inhibited leukocyte rolling at 4-24 h in wild-type animals and also blocked the residual rolling seen in L-selectin-deficient mice. Blocking E-selectin function had no effect on leukocyte rolling flux at any time point in wild-type or L-selectin-deficient mice. Despite reduced rolling, leukocyte adhesion and emigration were not measurably reduced in the L-selectin-deficient mice in this vascular bed. In conclusion, leukocyte rolling is L-selectin-dependent post-Ag challenge with L-selectin and P-selectin sharing overlapping functions.

Animals↗
PubMed1998

Differential induction of P-selectin expression on platelets by two cell separators during plateletpheresis and the effect of gender on the release of soluble P-selectin.

BACKGROUND: Though a number of studies have elegantly characterized platelet activation during storage, less attention has been paid to the initial activation caused by different collection procedures. STUDY DESIGN AND METHODS: The effects of two blood cell separators on the initial activation of platelets were measured by flow cytometric analysis of P-selectin expression in 13 male donors on one cell separator (CS 3000 Plus) and 11 men and 9 women on the other (MCS 3P). In addition, the storage and release of soluble P-selectin (circulating P-selectin [cP-selectin]) by platelets were quantified, to determine whether the change in cP-selectin is a more sensitive marker for initial platelet activation, and the influence of gender on measured endpoints was evaluated. RESULTS: The CS 3000 Plus increased the percentage of P-selectin-positive platelets from a median of 3.4 percent before apheresis to 7.6 percent (p = 0.006) in platelet concentrates (PCs), whereas the MCS 3P did not (p = 0.002 between the two cell separators). When preapheresis cP-selectin levels were compared to those in apheresis PCs, cP-selectin increased from 51 to 101 ng per mL in plasma of CS 3000 Plus PCs, whereas cP-selectin levels increased from 53 to 78 ng per mL in MCS 3P PCs (men) and from 48 to 99 ng per mL in MSC 3P PCs (women) (p<0.005 for all). The relative increase in cP-selectin was higher in women than in men in MCS 3P PCs (p = 0.013). Concomitantly, the amount of P-selectin stored in platelets before apheresis decreased (p<0.025 for all). When donors undergoing apheresis on the MCS 3P were compared, the amount of P-selectin stored in the platelets of PCs was higher in men than women (p = 0.026). CONCLUSION: This trial shows 1) that initial activation of platelets obtained with the MCS 3P is less than that of platelets obtained with the CS 3000 Plus; 2) that the increase in cP-selectin is a more sensitive marker for initial platelet activation than the expression of P-selectin on the surface; and 3) that the relative amount of cP-selectin is higher in women than in men given the same stimulus. Differences in platelet activation by various cell separators and the sex of the donor may contribute to variability of PC quality.

Blood Platelets↗
PubMed2006

Selectin ligand expression regulates the initial vascular interactions of colon carcinoma cells: the roles of CD44v and alternative sialofucosylated selectin ligands.

Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of approximately 170 and approximately 130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis.

Animals↗
PubMed2002

Platelet P-selectin levels in relation to plasma soluble P-selectin and beta-thromboglobulin levels in atrial fibrillation.

BACKGROUND AND PURPOSE: The increased risk of stroke and thromboembolism in atrial fibrillation (AF) may be related to a prothrombotic or hypercoagulable state, with abnormalities of hemostasis and platelet activation. To investigate the role of platelets in AF and the influence of antithrombotic therapy, we developed and then applied a new assay to detect the absolute amount of P-selectin per platelet (pP-selectin) based on cell lysis. Thus, pP-selectin in AF patients was compared with that of healthy controls and also with plasma soluble P-selectin (sP-selectin) and beta-thromboglobulin as established indices of platelet activation. METHODSMDSAH: We studied 122 patients (mean [SD] age, 71 [9] years; 65 men) with chronic AF of >6 weeks' duration: 34 were not on antithrombotic therapy, 30 were taking aspirin (75 to 300 mg/d), and 58 were fully anticoagulated with warfarin. pP-selectin was compared with sP-selectin and plasma beta-thromboglobulin levels (enzyme-linked immunosorbent assay). Results were compared with those of 23 healthy controls (mean [SD] age, 74 [9] years; 7 men) in sinus rhythm. RESULTS: pP-selectin was significantly lower in AF patients on no antithrombotic therapy (P=0.03) than in healthy controls, but sP-selectin and beta-thromboglobulin levels were not significantly different and did not differ in patients taking aspirin or warfarin. However, pP-selectin was lower in patients with AF on aspirin than in those on warfarin (P<0.05). pP-selectin/sP-selectin correlated significantly in healthy controls (r=0.47, P=0.03) but inversely (r=-0.43, P=0.03) in AF patients on no antithrombotic therapy. CONCLUSIONS: Lower levels of pP-selectin may represent a depletion of pP-selectin after platelet activation in AF. Aspirin further decreases pP-selectin levels compared with warfarin. On the basis of the principle of platelet lysis, we demonstrate that it is possible to determine the amount of P-selectin per platelet, which may be regulated in the megakaryocyte through a cyclooxygenase-dependent pathway.

Aged↗
PubMed1999

P-selectin deficiency exacerbates experimental glomerulonephritis: a protective role for endothelial P-selectin in inflammation.

P-selectin is a leukocyte adhesion receptor present in endothelial cells and platelets. We examined the role of P-selectin in the autologous phase of an accelerated model of anti-glomerular basement membrane (GBM) glomerulonephritis using P-selectin-deficient mice and chimeric mice expressing P-selectin only in platelets or endothelial cells. P-selectin-deficient mice exhibited more severe glomerular damage with increased interstitial mononuclear leukocytic infiltrates, and had significantly increased proteinuria and mortality when compared to wild-type mice. P-selectin on the endothelium was predominantly responsible for protection from the exacerbated disease, because chimeric mice with endothelial P-selectin, and not mice with platelet P-selectin, showed glomerular injury similar to that in wild-type animals. Levels of soluble circulating P-selectin were increased in nephritic wild-type mice and in chimeric mice with endothelial P-selectin, but not platelet P-selectin. Levels of soluble P-selectin, which has been shown to be anti-inflammatory in vitro, were inversely associated with the severity of disease. P-selectin was not expressed in the endothelium of the glomerulus or interstitium. Thus, the protective effect in wild-type mice may be accounted for, in part by soluble P-selectin shed by non-renal endothelial cells, although other endothelial P-selectin-dependent mechanisms cannot be ruled out.

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